Assembly of the Auditory Circuitry by a
Hox
Genetic
Network in the Mouse Brainstem
Maria Di Bonito1,2,3, Yuichi Narita4, Bice Avallone5, Luigi Sequino6, Marta Mancuso1, Gennaro Andolfi1, Anna Maria Franze`7,8, Luis Puelles9, Filippo M. Rijli4,10*, Miche`le Studer1,2,3*
1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy, 2 Universite´ de Nice-Sophia Antipolis, Nice, France, 3 INSERM UMR 1091, Nice, France, 4 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland,5 Department of Biological Sciences, Universita` degli Studi di Napoli Federico II, Naples, Italy, 6 Institute of Audiology, University ‘‘Federico II’’, Naples, Italy,7 Institute of Genetics and Biophysics ‘‘A. Buzzati Traverso’’ C.N.R., Naples, Italy, 8 CEINGE Biotecnologie Avanzate, Naples, Italy,9 Department of Human Anatomy and Psychobiology, University of Murcia, Murcia, Spain, 10 University of Basel, Basel, Switzerland
Abstract
Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.
Citation: Di Bonito M, Narita Y, Avallone B, Sequino L, Mancuso M, et al. (2013) Assembly of the Auditory Circuitry by a Hox Genetic Network in the Mouse Brainstem. PLoS Genet 9(2): e1003249. doi:10.1371/journal.pgen.1003249
Editor: Sabine Cordes, Samuel Lunenfeld Research Institute, Canada
Received February 29, 2012; Accepted December 2, 2012; Published February 7, 2013
Copyright: ß 2013 Di Bonito et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Telethon Foundation grant number TGM06AO3, Italy, and the ANR ‘‘2009 Chaire d’Excellence’’ Program, France, grant number R09125AA to MS; by the Spanish BFU2008-04156 and 04548-GERM-06 grants to LP; and by the Novartis Research Foundation, ARSEP, and the Swiss National Science Foundation (Sinergia Grant CRSI33_127440) to FMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist. * E-mail: filippo.rijli@fmi.ch (FMR); Michele.STUDER@unice.fr (MS)
Introduction
The mammalian brainstem plays a crucial role in the regulation of many vital functions through a complex system of reflex arcs and relays information to higher brain centers through inter-connected neuronal circuits. During development, the hindbrain becomes subdivided along the antero-posterior (A-P) axis into serially repeated, spatially segregated, modules of progenitor cells, the rhombomeres (r). Individual rhombomeres give rise to distinct portions of sensory and motor columns depending on the position of progenitors along the dorso-ventral (D-V) axis, respectively [1], thus generating nuclei of multi-segmental origin and topographic patterns of connectivity [2,3,4,5,6,7]. For instance, in the somatosensory system, afferent innervation from mandibular or whisker pad (maxillary) facial dermatomes targets the r2- or r3-derived components of the principal trigeminal sensory nucleus, respectively. In turn, the information is somatotopically relayed to the thalamus and somatosensory cortex, contributing to build a facial somatosensory map [7]. Vestibular nuclei also originate from different rhombomeres and display specific sets of axonal trajectories with distinct targets [3,4,8,9]. Thus, regional pattern-ing along the A-P axis and specific D-V determinants intersect to determine sub-circuit connectivity within functionally related longitudinal neuronal columns.
Topographic connectivity and employment of sensory and motor nuclei are also well described during formation of auditory-dependent circuits [10]. The auditory central pathway consists of sensory nuclei transmitting ascending acoustic information, and efferent motor neurons modulating primary afferent responses. The sensory organ for sound is the cochlea, which contains two types of receptors, namely the inner and outer hair cells. While the inner hair cells (IHCs) are the major detectors of auditory stimuli, the outer hair cells (OHCs) enhance low level sounds by increasing the amplitude and frequency selectivity of basilar membrane vibrations, a process called ‘‘cochlear amplification’’ [11,12]. From the periphery, sound information travels through the VIIIth cranial nerve to the brainstem cochlear nuclear (CN) complex, which is the primary relay station for central auditory processing [13]. This cochlear complex originates from distinct portions of the r2–r5 region, which will give rise to the anteroventral (AVCN), posteroventral (PVCN) and dorsal (DCN) cochlear nuclei, as well as to the cochlear granule cell population of the microneuronal shell [6]. A significant portion of the CN complex derives from a dorsal rim of neuroepithelium referred to as the auditory lip [6,14], which is part of the lower rhombic lip and selectively expresses the transcription factor Atoh1 (also known as Math1) [15,16,17]. Variously processed sound-related signals, ultimately leading to qualitative sound perception, travel from the cochlear nuclei through the lateral lemniscus (LL) complex to the inferior
colliculus (IC; midbrain) and medial geniculate nucleus (MG) of the thalamus, which in turn relays auditory information to the auditory cortex. On the other hand, temporal and spatial sound localization are relayed by the cochlear nuclei through a parallel pathway in the ventral brainstem, before reaching high level brain structures [18]. This pathway includes the superior olivary complex (SOC), which is mostly derived from r5 and is partly composed of the corresponding Atoh1+lineage [3,16,19].
Proper hearing function is also controlled by centrifugal (efferent) motor connections, which modulate the incoming afferent sensory auditory information. The major component is represented by the olivocochlear neurons (OC), a subpopulation of the inner ear efferent (IEE) neurons, which are born in ventral r4, cross the midline during early development and segregate from their vestibular counterpart around embryonic day 14.5 of gestation (E14.5) in mice [20,21]. While lateral OC (LOC) motor neurons innervate afferent sensory neurons in contact with the IHCs, modulate cochlear nerve excitability and protect the cochlea from neuronal damage in acute acoustic injury [21,22], medial OC (MOC) motor neurons are innervated by reflex neurons of the PVCN [23,24] and regulate the vibrating OHCs in the cochlea, modulating in this way the ‘‘cochlear amplification’’ process [25,26,27]. This is known as the MOC reflex. Cochlear efferent motor neurons also play a role in the normal maturation of afferent responses, particularly during the early postnatal period [21,28]. Another feedback reflex, the middle-ear muscle reflex (MEM), is closed by facial and trigeminal branchiomotor neurons (FBM, TBM) that activate the stapedius and tensor tympani muscles respectively, thus tensing the chain of tympanic ossicles and reducing the amplitude of sound transmission through the middle ear [29,30]. Thus, the MOC and MEM reflexes represent two parallel sound-evoked feedback mechanisms acting on the auditory periphery to modulate incoming acoustic stimuli [29,31,32].
Little is known about the molecular determinants involved in the assembly of rhombomere-derived auditory sub-circuits. The Hox genes, a large family of homeobox-containing genes, display rhombomere-restricted expression patterns and provide early
patterning information to progenitors and their neuronal deriva-tives [33,34]. In turn, the expression of several Hox genes is maintained through later stages of circuit formation in distinct, rhombomere-derived neuronal subpopulations contributing to portions of hindbrain sensory and motor nuclei [4,7,35,36]. In the developing hindbrain, Hoxb1 selectively expressed in r4 is required to pattern r4-derived ventral efferent neurons, such as IEE and FBM, and to maintain normal levels of Hoxb2 in r4 [37,38]. Hoxb2 and Hoxa2, unlike Hoxb1, are expressed in r2 to r5 auditory derivatives. Moreover, the expression of Hoxb2 and Hoxa2 is maintained in the ventral CN, ventral nucleus of LL (VLL), and SOC nuclei throughout prenatal and postnatal developmental stages [36]. Thus, Hox genes are prime candidates to be involved in the assembly of sensorimotor functional circuits in the developing hindbrain.
In this study, we find that rostral rhombomeres and their associated Hox genes are required in establishing and maintaining two major functional circuits in the central auditory system, which have different rhombomeric origins. Firstly, we carried out a detailed fate map of r4 derivatives by generating a novel, highly restricted, r4-specific Cre-recombinase driver. We show that cells originating from r4 significantly contribute to the VLL, an important relay station in the sound perception pathway. Furthermore, we found that r4 largely supplies cells to the PVCN and DCN, a finding largely in agreement with previous work [6,16], but not to the granule cells of the microneuronal shell. Secondly, in Hoxb1 and Hoxb2 mutants, the VLL, PVCN, and MOC motor neurons are selectively affected, though with different severities, leading ultimately to elevated auditory thresholds in adult mutant mice. Thirdly, Hoxb1 negatively modulates Hoxa2 during r4 patterning, whereas Hoxa2 is mainly required in r2/r3 AVCN-derived development. Moreover, early conditional Hoxa2 inactivation in rhombic-lip derivatives selectively perturbs the AVCN axonal pathfinding to the medial nucleus of the trapezoid body (MNTB), resulting in decreased contralateral and increased ipsilateral targeting of MNTB due to the down-regulation of Rig1, the main axon guidance receptor for midline crossing. Altogether, this study provides, for the first time, genetic and functional evidence for a Hox gene network in the establishment and maintenance of proper auditory rhombomere-specific circuitry during hindbrain development.
Results
Mapping of rhombomere 4-specific contribution to the auditory system
Taking advantage of specific r2- and r3/5-Cre-expressing mouse lines [39,40], previous studies have genetically mapped the contribution of the r2–r5 rhombic lip to distinct portions of the CN and SOC complexes [6,16,19]. However, previous attempts to generate r4-specific lines invariably resulted in additional Cre expression caudal to r4 [7,16,41]. In this study, we generated a novel mouse transgenic line, named b1r4-Cre, which allowed us to exclusively map r4 and its derivatives throughout the mature brainstem (Figure 1A and Figure S1). To this purpose, we used a well characterized enhancer from the Hoxb1 locus [42] to drive the Cre-recombinase gene exclusively in r4 (Figure S1A). In b1r4-Cre transgenic animals, onset of Cre expression in presumptive r4 occurs first in a mosaic fashion (Figure S1C), but from E9.0 onwards, Cre is expressed throughout r4 as shown in Figure S1D. To permanently label the polyclonal population of cells derived from r4, the b1r4-Cre transgenic line was mated to the ROSA26-YFP reporter mouse [43] and progenies positive for both alleles (herein called b1r4-Cre/YFP)
Author Summary
Sound perception and sound localization are controlled by two distinct circuits in the central nervous system. However, the cellular and molecular determinants un-derlying their development are poorly understood. Here, we show that a spatially restricted region of the brainstem, the rhombomere 4, and two members of the Hox gene family, Hoxb1 and Hoxb2, are directly implicated in the development of the circuit leading to sound perception and sound amplification. In the absence of Hoxb1 and Hoxb2 function, we found severe morphological defects in the hair cell population implicated in transducing the acoustic signal, leading ultimately to severe hearing impairments in adult mutant mice. In contrast, the expression in the cochlear nucleus of another Hox member, Hoxa2, regulates the guidance receptor Rig1 and contralateral connectivity in the sound localization circuit. Some of the auditory dysfunctions described in our mouse models resemble pathological hearing conditions in humans, in which patients have an elevated hearing threshold sensitivity, as recorded in audiograms. Thus, this study provides mechanistic insight into the genetic and functional regulation of Hox genes during development and assembly of the auditory system.
Figure 1. Rhombomere 4 neuronal derivatives contribute to nuclei involved in auditory perception. (A) Strategy for the Cre-loxP recombinase r4-fate map. Upon Cre-mediated recombination, the loxP sites surrounding the PGKneo cassette of the ROSA26 YY reporter line are excised and YFP is expressed exclusively in r4 and r4 derivatives. (B) Dorsal and lateral views of a E10.5 b1r4-Cre/YFP embryo show restricted expression of YFP in r4 and neural crest-derived cells (ncc) in the second branchial arch (ba2). The white line delineates the level and plane of sagittal section of panels in (C). (C) r4-restricted immunostaining of Cre-recombinase in progenitors of the ventricular zone (vz). YFP+/Cre2post-mitotic cells at
the marginal zone (mz) originate from YFP+/Cre+progenitors. (D) Sagittal sections of an E12.5 b1r4-Cre/YFP embryo immunostained with a GFP antibody reveal the r4 domain, the caudal migration of facial branchiomotor neurons (mFBM), the ventricular to pial migration of presumptive lateral lemniscus cells (mVLL) and the lateral lemniscus tract (LLt) projecting rostrally. Below, a schematic of an E14.5 sagittal section indicating the position of the various nuclei. The red line delineates the plane of section of panels (E). (E) The olivocochlear (OC) neurons (delimitated by a red contour) express choline acetyltransferase (ChAT), Gata3 and Tbx20. (F) Schematic coronal section of a P8 brain illustrating the positions of the various nuclei. The lateral superior olive (LSO) but not the medial superior olive (MSO) nucleus, has an r4 origin, as indicated by YFP and VGlut2 staining. ChAT and Tbx20 are expressed in lateral (LOC) and medial OC (MOC) neurons within the LSO and ventral to the LSO, respectively. (G) Sagittal sections at different ages indicating the YFP+r4-derivatives: the ventral lateral lemniscus (VLL) (rostrally) and FBM (caudally) nuclei. VLL and cochlear neurons project rostrally to the inferior colliculus (IC) and some fibers continue to the thalamus (arrowheads). (H) Coronal sections of P0 pups indicate high contribution of r4/YFP+cells to the VLL, positive for Gad67, but not to the dorsal LL (DLL), which is VGlut2+. Hoxb2 and Hoxa2 are expressed in the VLL and pontine nuclei (PN). A few dispersed YFP+cells are identified in the PN. (I) Schematic of a P8 brain sagittal section showing the position of the cochlear nuclear complex (CN) and its subdivision into anteroventral (AVCN), posteroventral (PVCN) and dorsal (DCN) nuclei. Adjacent sagittal sections illustrate a high contribution of YFP+cells to the PVCN and DCN. The arrowhead indicates the origin of r4-migrating cells. Dots delineate the presumptive boundary between AVCN and PVCN. Only a few YFP-positive cells label the AVCN, which is highly positive for Atoh7. The small intensely basofilic granule cells confined to the microneuronal shell (mnsh and outlined) and identified by Nissl and Pax6 expression (J) are not positive for YFP, indicating that cochlear granule cells do not have an r4-origin. (K) Similarly, YFP+cells do not co-localize with calbindin- (CB) and calretinin- (CR)
expressing cells in the PVCN region. (L) Schematic of the hindbrain in which rhombomeres 2 to 5 and their respective Hox genes are color-coded. The same code refers to (M). (M) Overview of the two main central auditory pathways, the MOC reflex and their rhombomeric origin. While r4-derivatives (in red) contribute mainly to the ascending sound perception pathway, which runs from the CN through the VLL to the IC, r2-, r3- and r5-derivatives (in green) contribute mainly (but not exclusively) to the pathway running through the superior olivary complex (SOC), which function in the localization of the temporal and spatial origins of sounds. The MOC reflex comprising of sensory PVCN interneurons and motor efferent OC neurons is also an r4-derivative. Vn, trigeminal motor nucleus; MNTB, medial nucleus of trapezoid body; Pr5 principal sensory trigeminal nucleus. Scale bars, 100 mm (C), 200 mm (D–J left), 20 mm (J right, K). See also Figures S1 and S2.
were analyzed (Figure 1A). Accordingly, at E10.5 activation of YFP was observed exclusively in r4 and its cellular progeny, as shown by double staining of Cre and YFP (Figure 1B, 1C). No ectopic expression of Cre was detected at later stages (data not shown).
We next analyzed the b1r4-Cre/YFP mouse line at different embryonic stages and early postnatal ages, using an anti-GFP antibody that cross-reacts with the YFP protein and labels proliferative and migrating/differentiating cells and their axonal projections. The r4 radial histogenetic territory itself is massively labeled and becomes morphogenetically deformed into a wedge-shaped, dorsally compressed configuration (Figure 1D, 1G). The first cohort of cells migrating out of r4 consists of the well-described caudomedial stream of tangentially migrating FBM neurons, which first move caudolaterally into r6 and then reach their definitive ventrolateral subpial position by radial migration [44,45,46] (Figure 1D, 1G). Another r4-derived efferent popula-tion is represented by OC neurons [20,21], a subpopulapopula-tion of IEE neurons. At E14.5 they form a compact superficial group of cells at the r4/r5 margin and selectively express the cholinergic marker ChAT [21] and the transcription factors Gata3 and Tbx20 [47,48] (Figure 1E). At P8 the OC neurons split into lateral (LOC) and medial (MOC) components [49], which express ChAT and Tbx20 and become located within the lateral superior olive (LSO) and in the medioventral portion of the SOC as scattered cells, re-spectively (Figure 1F). A part of the LSO, expressing the glutamatergic marker VGlut2 [16], is included within the r4 domain (Figure 1F).
Another sizeable stream of labeled r4-derived cells, which was not previously described, migrates to the basal longitudinal zone and then moves rostrally along the growing lateral lemniscus tract, which courses obliquely through the rostral hindbrain (Figure 1D, 1G). These cells will contribute to the majority of the VLL from E14.5 onwards (Figure 1G). At E16.5, labeled ascending lateral lemniscus fibers, originating from the ipsilateral r4-derived VLL neurons and also from the r4-derived projection neurons of the contralateral CN [50], reach the IC. At E18.5, lateral lemniscus fibers also extend along the brachium of the IC into the medial geniculate nucleus of the thalamus, and collaterals can be distinctly seen in the superior colliculus intermediate layers at P8 (Figure 1G, and data not shown). Transverse sections at P0 clearly show a high density of YFP+cells in the VLL but not in the dorsal nucleus of LL (DLL), which expresses the glutamatergic marker VGlut2 and originates from the Atoh1+lineage [19,51]. The majority of VLL neurons express the inhibitory GABAergic/glycinergic marker Gad67, a particular feature of this auditory structure, as previously described [52], and Hoxb2 and Hoxa2 [36]. In summary, our fate map study shows for the first time that r4 massively contributes to the VLL within the LL complex.
Next, we mapped precisely r4 contribution to the pluriseg-mental CN complex (Figure 1I). Previously, the contribution of r4 was indirectly inferred from the mapping of r3 and r5 derivatives, or from the mapping of the territory posterior to r3 [6,16]. These studies indicated that the AVCN is derived from r2 and r3, the PVCN from r3 and r4, and the DCN from r4 and r5 (summarized in Figure 1L, 1M). A significant proportion of these nuclei were strongly affected in Atoh1 conditional and null mutants, supporting their origin from the Atoh1+auditory lip [16,17].
Our results are largely in agreement with previous data and further extend them. First, we show that at E10.5 the YFP+ domain includes Atoh1-expressing cells in the rhombic lip region of dorsal r4 (Figure S2A). However, at E14.5 when Atoh1+ cells migrating from r2 to r5 rhombic lip invade the presumptive cochlear complex [17], only a few r4-derived YFP+cells overlap
with Atoh1 expression domain, whereas no co-localization of YFP with the granule cell marker Barlh1 [6] is found in this region (Figure S2B). Secondly, we show that YFP+cells contribute to an intermediate sector across the CN complex, which crosses dorsoventrally the magnocellular core portion of the DCN and then gives rise to the majority of the PVCN (Figure 1I and Figure S2C–S2E). Small portions of the DCN remain unlabeled, suggesting additional contribution from r5, as previously reported [6,16], but also a likely contribution from r3 to the region of DCN anterior to the YFP+domain. Thirdly, we found that only a small number of YFP+cells are distributed in the AVCN at P8, which, unlike the PVCN, expresses high levels of Atoh7 (also known as Math5) [53] (Figure 1I). Finally, we started to characterize the cellular identity of YFP+cells and found that at P8, r4-derived cells fail to co-express Pax6 (Figure 1J), a marker for the microneuronal granule cell population [15,54]. Moreover, YFP signal is absent in calbindin- and calretinin-expressing neurons (Figure 1K), corre-sponding to octopus and globular-bushy cells, respectively [55,56]. These data indicate that subpopulations of glutamatergic neurons, which normally derive from the Atoh1+ neuroepithelial domain [15], do not originate from r4. A full characterization of YFP+cells in embryonic and adult stages will be reported elsewhere (M.D., L.P. and M.S., in preparation).
In summary, we show that r4 largely contributes to the motor cochlear efferent neurons, to the relay VLL neurons, and within the cochlear nucleus, to the majority of the PVCN, and part of the DCN. Thus, while r4 seems to be required for the structures involved in sound transmission, amplification and protection (i.e. PVCN, VLL, and OC), r2, r3 and r5 are likely contributing to components of the sound localization pathway that runs through the SOC and trapezoid body complex before reaching higher-order auditory structures (Figure 1L, 1M).
Overall, our r4 fate mapping, combined with previously published r2, r3 and r5 maps [6,16] and patterns of early and late Hoxa2 and Hoxb2 expression profiles [36], strongly suggest rhombomere-specific and Hox-dependent assembly of the two main central auditory sub-circuits (Figure 1L, 1M).
Regulation of Hoxb1, Hoxb2, and Hoxa2 in sensory r4 of Hox mutant mice
Previous work dissected the genetic and regulatory network involved in establishing and maintaining the identity of r4 progenitors [34]. Hoxb1 plays a key role in patterning ventral r4 progenitors, partly through transcriptional regulation of Hoxb2 and Hoxa2 [37,38,57]. Maintenance of Hoxb1 expression in ventral r4 requires both Hoxb1 itself and Hoxb2 through auto- and cross-regulatory interactions, respectively [37,58,59,60]; however, it is not known whether a similar mechanism is acting in dorsal/ sensory r4. Unlike Hoxb1, Hoxb2 and Hoxa2 expression is maintained in differentiated r4-derivatives, such as the VLL and VCN ([36] and our study). Thus, Hoxb1 may pattern sensory r4-derivatives by regulating dorso-ventral Hoxb2/Hoxa2 expression in r4 progenitor cells, hence controlling post-mitotic specification during the development of the sensory system. To test this hypothesis and investigate the roles of Hoxb1, Hoxb2, and Hoxa2 in the development of auditory pathways, we analyzed the effects of their functional inactivation.
Hoxb1 functional deletion results in an early re-patterning of the ventral r4 territory into a more anterior identity [44,61]. To bypass the early Hoxb1 role in r4 neuroepithelium and investigate its requirement during neurogenesis, we generated a novel Hoxb1 conditional mutant allele, Hoxb1flox (Figures S3 and S4; see Materials and Methods) and mated it to the b1r4-Cre driver. Conditional b1r4-Cre;Hoxb1flox/floxhomozygous mutant mice
after referred to as Hoxb1lateCKO) are viable and fertile. Mutant embryos retain Hoxb1 expression in r4 until E8.75–E9.0 (Figure 2A), in accordance with the timing of Cre expression and onset of Cre-mediated excision (Figure 1 and Figure S1). At E9.25, Hoxb1 expression is no longer present in mutant r4, while the remainder of its expression is unaltered (Figure 2A).
We also obtained constitutive Hoxb1null homozygous mutants (Figures S3 and S4; see Materials and Methods) that exhibit total loss of Hoxb1 expression from its onset (Figure 2A) and lack any selectable cassette that might interfere with adjacent Hox genes [62,63]. To specifically trace r4 derivatives from the control and the two distinct Hoxb1 mutants throughout embryonic and postnatal brains, Hoxb1flox and Hoxb1null homozygous mice were mated with the b1r4-Cre/YFP reporter line (see Materials and Methods for mating schemes). In addition, we generated a novel, viable and fertile Hoxb2 homozygous mutant allele (hereafter referred to as Hoxb2DKO), that, similarly to the Hoxb1nulland unlike previously described Hoxb2 knockout alleles [37,58,64], has no selectable marker left within the locus (see Materials and Methods). Finally, we made use of the previously described Hoxa2null and conditional Hoxa2floxalleles [7,65].
Next, we assessed Hox cross-regulatory interactions within r4 in Hoxb1, Hoxb2 and Hoxa2 mutant alleles. Flat-mounted prepara-tions and sagittal secprepara-tions of control embryos show high Hoxb2 expression levels in r4 to r6 and low levels in r3 (Figure 2B). In E10.5 Hoxb1null and Hoxb1lateCKO hindbrain preparations, Hoxb2 expression is down-regulated throughout r4 to similar levels as in r3 (asterisks in Figure 2B), resulting in a duplication of r3-features (‘‘r3’’ in Figure 2B). This is confirmed in mid-sagittal sections showing a decrease of Hoxb2 expression in r4 progenitors of Hoxb1null and Hoxb1lateCKO. Expression is still maintained in early differentiating cells, similarly to r3 (arrowheads in Figure 2B). In contrast, Hoxa2 is normally expressed at low levels in r2 and r4, and at high levels in r3, particularly in a wide intermediate stripe of the dorsal sensory column (horizontal bracket in Figure 2C) and in a thinner stripe laterally (vertical bracket in Figure 2C), the presumptive auditory column. In E10.5 Hoxb1 mutant embryos, expression of Hoxa2 is abnormally up-regulated in r4, pre-dominantly in the two sensory stripes, resulting in a duplication of r3-typical features in r4 (Figure 2C). Sagittal sections at different alar plate levels of Hoxb1nullmutant embryos confirm up-regulation of Hoxa2 in the ventricular zone, and, strikingly, also in the mantle zone of r4 (arrows in Figure 2D and inset), which normally expresses low levels of Hoxb1 and Hoxa2 (arrowheads in Figure 2D and inset). In mutant r4, Hoxa2 is maintained at high levels in the post-mitotic neurons, similarly as in r3. Thus, our data show that in the absence of Hoxb1, r4 acquires Hox features typical of r3, such as low levels of Hoxb2 and high levels of Hoxa2, indicating a re-patterning of r4 into r3. In addition, Hoxb1 differentially regulates Hoxb2 and Hoxa2 expression levels in r4, further supporting a complex regulatory interaction between Hoxb1 and Hoxb2/Hoxa2 in specifying r4 identity [37,38,57,58,59].
To investigate whether Hoxb2 acts similarly to Hoxb1 in r4 patterning, we assessed Hoxb1 and Hoxa2 expression in WT and Hoxb2DKOhindbrains from E8.75 to E10.5. Hoxb1 protein is lost in Hoxb2DKOat E10.5, though it is present at earlier embryonic stages (Figure 2E), indicating that Hoxb2 is required for maintaining Hoxb1 in r4. Similarly to Hoxb1 mutants, Hoxa2 expression is dramatically up-regulated throughout r4, with particular emphasis in the intermediate and lateral columns (Figure 2F), and in post mitotic neurons (arrows in Figure 2G and inset). In contrast, no changes are observed in Hoxa2nullembryos (Figure 2H), apart from the r2/r3 alar defects previously described [66]. Taken together, our data show that within r4, Hoxb2 acts mainly in maintaining
high expression of Hoxb1 and that in its absence, r4 acquires tr3-typical features, similarly to Hoxb1 mutant hindbrains. Importantly, we found increased Hoxa2 expression, at levels similar to r3, in post-mitotic r4 neurons of Hoxb1 and Hoxb2 mutants, implying that r4-derived sensory cells abnormally maintain Hoxa2 expression throughout hindbrain development (Figure 2I).
Finally, we investigated whether Hoxb1 and Hoxb2 are required in the differentiation process of the IEE and OC motor neurons, which normally differentiate in ventral r4 and interact with dorsally-derived sensory structures during the development of the auditory sensorimotor circuitry. At E10.5, IEE are located in the r4 mantle zone next to the floor plate and normally express Gata2/ 3, Isl1 and Phox2b [48,59,67] (Figure S5A, S5B). No IEE neurons are identified in E10.5 Hoxb1nulland Hoxb1lateCKOembryos, as seen by the absence of Gata2/3 expression within the pool of Phox2b+/ Isl1+motor neurons (Figure S5B). This is not due to a delay in specification, since no OC neurons, positive for ChAT and Gata3, located in ventral peri-olivary positions can be distinguished in E14.5 Hoxb1 mutant embryos (Figure S5E). On the contrary, a few Gata3+ and Tbx20+ cells are preserved in Hoxb2DKO mutant embryos at E10.5 and E14.5, even if they are located in a slightly more dorsal position than in control embryos (Figure S5C, S5F). These data indicate that early IEE and late OC specification are maintained in Hoxb2DKO embryos despite the late absence of Hoxb1 in r4.
Hoxb1 is a key determinant gene in r4-derived VLL development
To investigate the requirement of Hox genes in the generation of sensory auditory structures, we first analyzed the size and position of the VLL with various markers on adjacent coronal sections at E18.5 and sagittal sections of P8 WT and mutant brains (Figure S6 and Figure 3). Very few cells contributing to the VLL are identified in E18.5 Hoxb1nullmutant hindbrains, as shown by YFP and Gad67 staining (Figure S6B). Only some YFP+ cells with lemniscal projections, scattered rostral to the r4 wedge, are still maintained in Hoxb1null brains at P8 (Figure 3A). No detectable Hoxb2 or Hoxa2 expression can be found in sagittal sections at all levels, whereas reduced expression of Gata3 and Gad67, which both label the GABAergic/glycinergic cellular cohort of the VLL [52,68], is still present in the remaining VLL, which is reduced by almost 90% in area (91.960.02) when compared to control VLL (Figure 3A, 3D). This indicates that the early absence of Hoxb1 function prevents normal r4-derived VLL specification and/or migration, and supports a major contribution of r4 to the formation of the VLL, particularly to its GABAergic cohort, which contributes to the majority of the VLL [52]. Moreover, absence of VGlut2 expression observed at E18.5, and confirmed at P8 (Figure S6B and Figure 3A), rules out any inhibitory to excitatory cell fate transformation within the VLL.
A less severe reduction of the VLL was observed in Hoxb1lateCKO mutants (n = 3; 49.260.1% in area as compared to WT; Figure 3D and legend). This was mainly supported by the maintenance of Gata3-, Gad67-, Hoxb2- and Hoxa2-expressing cells at E18.5 and
P8, and by the presence, although reduced, of YFP+ VLL
projections to the IC (Figure 3A and Figure S6B). Similarly, the VLL area is reduced by 41.760.3% in Hoxb2DKO (n = 3) when compared to WT (n = 3) brains (Figure 3D and legend), whereas no size reduction is measured in the VLL of Hoxa2null mutants (n = 3; 10565.7%) at E18.5 before Hoxa2nullperinatal death [69] (Figure 3C, 3E and legend). Together, these data indicate that Hoxb1 is an important determinant gene in r4-derived VLL development, because r4 to r3 change of identity occurring in
Figure 2. Regulatory interactions betweenHoxb1,Hoxb2, andHoxa2in r4. (A) The diagrams above the panels indicate the interactions between Hoxb1 and Hoxb2. While Hoxb1 auto-regulates its own expression in r4, it also binds to an Hoxb2 r4 enhancer to maintain Hoxb2 expression in r4. Hoxb2 maintains expression of Hoxb1 in r4. Crosses indicate loss of Hoxb1 protein in Hoxb1nullembryos and loss of the auto- and cross-Hox Genes in Central Auditory Circuits
Hoxb1 mutants prevent a proper VLL nucleus development. However, the persistence of some GABAergic cells in the VLL of Hoxb1nullbrains, suggests that other rhombomeres besides r4 might contribute to VLL formation. In contrast, Hoxb2 or Hoxa2 do not appear to play a predominant role in VLL specification. The reduction in VLL size in Hoxb2 mutant brains is most probably due to the failure of Hoxb2 maintaining Hoxb1 expression in r4, as suggested by the lack of statistically significant differences in VLL
size between Hoxb1lateCKOand Hoxb2DKO mutant animals
(Figure 3D).
Abnormal specification of r4-derived ventral cochlear structures in Hox mutants
We next investigated the involvement of Hoxb1, Hoxb2 and Hoxa2 in the development of the CN complex (Figure 4). In Hoxb1 mutant mice, no considerable changes in the overall size of the CN were observed at P8. However, careful analysis showed that r4-derived YFP+ cells massively invade the granule shell layer,
normally derived from the Atho1+ lineage [15,17,19], and
ectopically expressed Pax6, as confirmed by the abnormal presence of double YFP+/Pax6+cells in the microneuronal shell (Figure 4A). This strongly indicates that r4-derived mutant cells have now acquired a granule cell identity, which r4 does not normally contribute to (Figure 1I–1J and Figure 4A). In addition, while Hoxb2 expression is maintained in the increased microneur-onal shell of the Hoxb1 mutants, Hoxb2 and Hoxa2 expression levels are slightly affected in the ventral CN (Figure 4B). Namely, Hoxb2 is down-regulated in the PVCN (asterisks in Figure 4B), whereas Hoxa2 expression is slightly up-regulated in PVCN regions ventral to the microneuronal shell layer (arrows in Figure 4B), in line with their respective altered expression levels observed at E10.5 (Figure 2B–2D). Notably, Atoh7, predominantly expressed in specific Atoh1-derived post-mitotic glutamatergic populations of the AVCN [15,53], is now strongly up-regulated in the PVCN of Hoxb1nulland Hoxb1lateCKOmutants, indicating that the PVCN has acquired features of the r2/3-derived AVCN [6] (summarized in Figure 4E). Next, we asked whether Hoxb2 and Hoxa2 are also required in the specification of VCN components. Interestingly, Atoh7 and Hoxa2 expressions are also increased in the PVCN of Hoxb2DKO (arrows in Figure 4C). In contrast, Atoh7 and Hoxb2 expressions are strongly reduced in the AVCN of E18.5 Hoxa2null mutants (arrows in Figure 4D), in accordance with the early
patterning defect previously described in the rostral hindbrain of these mutants [66].
The dramatic increase of cells that express high levels of Atoh7 in the PVCN and the ectopic formation of YFP+ granule cells observed in Hoxb1 mutants (both are Atoh1+lineage derivatives), led us to hypothesize that an increase of Atoh1 in r4 is the cause of the ectopic generation of glutamatergic neurons. Normally, the Atoh1+domain in dorsal r4 (i.e. YFP+) of E10.5 embryos is smaller than in adjacent rhombomeres, such as r3 (Figure 4F). In contrast, in Hoxb1nullmutant embryos this domain is enlarged to a similar extent as in control r3, suggesting an up-regulation of Atoh1 in r4, which may contribute to the acquisition of r3-like fate and hence to the ectopic generation of glutamatergic cell types (summarized in Figure 4F). This is even more pronounced at E14.5, when cells from the r2 to r5 rhombic lip contribute to the CN primordium. In WT embryos, the majority of YFP+cells is identified lateral to the Atoh1+region, whereas YFP+cells abnormally express Atoh1 and clearly invade the Atoh1+ domain in Hoxb1null and Hoxb1lateCKO mutants, as demonstrated by the sizeable overlap of both domains (Figure 4G). Hence, Hoxb1 normally restricts the Atoh1+domain in r4, impinging in this way on a specific r4 dorsal fate, which is different from the Atoh1+ lineage-related ones of adjacent rhombomeres. In summary, these data show that Hoxa2 is involved in the formation of the r2/3-derived AVCN, whereas Hoxb1 and Hoxb2 are required in the specification of the r4-derived PVCN by imposing an r4-specific identity during auditory development.
Axon pathfinding defects of cochlear nuclei in Hox mutant mice
We next assessed whether deletion of specific Hox genes may have direct consequences on the VCN connectivity pattern at postnatal stages. In P8 Hoxb1null and Hoxb1lateCKO mutants, we identified ectopic r4-derived YFP+projections crossing the ventral midline and innervating the medial nucleus of the trapezoid body (MNTB), a normal contralateral target of r2/3 AVCN-derived fibers. These projections are never labeled by YFP in control mice, since they do not originate from the PVCN, the major source of r4-derived YFP+ CN projections (Figure 5A). Similarly, dextran injections in the PVCN of Hoxb2DKO label ectopic projections to the contralateral MNTB (cMNTB) (arrowhead in Figure 5B), whereas in control mice the very few axons projecting ventrally normally innervate contralateral MOC neurons as part of the
regulatory loops in Hoxb1lateCKOmutants. Lateral views of E8.5 to E9.25 embryos indicate that while Hoxb1 expression is still maintained in r4
(although at lower levels) of E8.75 Hoxb1lateCKOmutants, r4 expression is completely abolished in E9.25 mutant embryos (arrowheads). Expression in the posterior region is still maintained at both ages (arrows). (B) Ventricular views of flat-mount preparations of E10.5 WT, Hoxb1nulland Hoxb1lateCKO
hindbrains hybridized with Hoxb2. Expression of Hoxb2 is strongly decreased (but not abolished) in r4 of Hoxb1nulland Hoxb1lateCKOembryos, at similar levels to r3 (asterisks). R4 acquires an expression pattern of r3, as indicated by ‘‘r3’’. Down-regulation of Hoxb2 in r4 can also be appreciated in mid-sagittal sections of mutant embryos. The line of cells expressing high levels of Hoxb2 denotes early post-mitotic cells (arrowhead). (C) Ventricular views of flat-mount preparations of E10.5 WT, Hoxb1nulland Hoxb1lateCKOhindbrains hybridized with Hoxa2. Expression of Hoxa2 is increased in r4 and the characteristic Hoxa2 expression profile of r3 is now duplicated in r4 of Hoxb1nulland Hoxb1lateCKOembryos supporting an r4 to r3 change of
identity. The horizontal and vertical brackets indicate higher expression domains of Hoxa2, respectively in a large intermediate stripe and in a thinner lateral stripe of the sensory column, the presumptive auditory column. (D) On the left, expression of Hoxb1 indicates the position of r4 on sagittal sections. On the right, increased expression of Hoxa2 is detected in the ventricular and mantle layers of r4 at two different dorsal levels in Hoxb1null embryos. In mutant embryos, the expression of Hoxa2 is maintained at levels comparable to r3 in the r4 mantle zone (mz) (i.e. post mitotic neurons) with respect to WT (arrows, see also insets). (E) In Hoxb2DKOmutants, lack of Hoxb2 (indicated with a cross) results in failure to maintain Hoxb1 expression in r4. Sagittal and coronal views show that Hoxb1 protein is present in r4 of E8.75 embryos, but not maintained in E10.5 Hoxb2DKOmutant embryos. (F) Flat-mounted hindbrain preparations hybridized with Hoxa2 show a duplication of r3 features in r4 in E10.5 Hoxb2DKOmutant embryos.
Hoxa2 expression levels are increased in r4 in the absence of Hoxb2, similarly to Hoxb1 mutant embryos. (G) E10.5 sagittal sections in dorsal regions indicate increased expression of Hoxa2 in ventricular (vz) and mantle (mz) zones of r4 (arrows, see also insets) mimicking the expression profile of r3. (H) Flat-mounted hindbrain preparations of E10.5 WT and Hoxa2nullembryos hybridized with Hoxb2. No particular expression changes can be observed in mutant embryos. Red arrowheads indicate a defect in alar r2/r3, as previously described [66]. (I) Schematics summarizing the expression of Hox genes in r4 of Hoxb1 or Hoxb2 mutants. Scale bars, 200 mm (B, C, F and H); 100 mm (E top); 50 mm (E bottom). ba2, second branchial arc; me, mesoderm. See also Figures S3, S4 and S5.
Figure 3. The r4-derived VLL is affected inHoxb1andHoxb2mutant mice. (A) Schematic view of a sagittal brain section indicating the YFP+
r4-derived nuclei and projections. A strong reduction of the YFP+VLL nucleus (arrowhead) and projections (arrow) in Hoxb1 mutants is observed. In constitutive mutants the reduction is much more severe than in conditional mutants, as quantified in (D). Adjacent sagittal sections show no Hoxb2 and Hoxa2-expressing cells in Hoxb1nullmutants, whereas cells in the reduced VLL of Hoxb1lateCKOstill express Hoxb2 and Hoxa2. Adjacent sections of another P8 pup confirm reduction of the VLL and indicate persistence of Gata3- and Gad67-expressing cells in both Hoxb1 mutants. No ectopic expression of VGlut2 is detected in the VLL region. (B) The VLL is reduced in Hoxb2DKOmutant pups, similarly to Hoxb1lateCKOmutants, as indicated by expression of Gata3, Gad67, Hoxa2 and quantification in (D). (C) In contrast, Hoxa2nullmutants show no significant changes in the VLL position and
size quantified in (E). The apparently bigger shape is due to the slightly oblique sections in mutant compared to WT brains. (D) Histogram showing the percentage of the VLL area size in WT (set up to 100%) and in the different genotypes as indicated on the y-axis. Mutants show statistically significant differences when compared to WT, or when Hoxb1lateCKOand Hoxb2DKOare compared to Hoxb1null(inter-genotype comparison, ANOVA
p,0.001; Hoxb1nullversus WT: p = 0.001; Hoxb1lateCKO versus WT: p = 0.01; Hoxb2DKO versus WT: p = 0.04; Hoxb1lateCKOversus Hoxb1null: p,0.001; Hox Genes in Central Auditory Circuits
MOC reflex (arrow in Figure 5B). This is in keeping with the finding that Atoh7 is increased in the PVCN of Hoxb1null, Hoxb1lateCKO and Hoxb2DKO mutants (Figure 4B, 4C), and the
notion that AVCN Atoh7+ neurons normally target the MNTB
[10,53]. Thus, the molecular identity transformation of PVCN to AVCN observed in Hoxb1 and Hoxb2 mutant mice is further supported by abnormal connectivity to their respective targets (Figure 5C).
To directly investigate the role played by Hoxa2 in VCN connectivity, we crossed the Hoxa2floxallele with a Wnt1::Cre driver [70] that allowed cell-autonomous inactivation of Hoxa2 at early stages in rhombic lip (and neural crest) derivatives. Wnt1::Cre;Hox-a2flox/flox mutants die around birth due to impaired neural crest development [71], thus preventing postnatal analysis of cochlear nuclei axon connectivity and functional impact on auditory function. Nonetheless, anterograde tracing by dextran injection in the AVCN of control and Wnt1::Cre;Hoxa2flox/floxbrains at E18.5 reveal a neuronal population aberrantly innervating the ipsilateral MNTB (iMNTB) in mutant brains (arrowhead in Figure 5D). This phenotype is reminiscent, though less prominent, of the defects observed in the Robo3/Rig1 mutant mice, in which AVCN projections are prevented from crossing the midline and accumu-late ipsiaccumu-laterally [72]. Analysis in E13.5 control and Hoxa2 conditional mutant mice revealed that Rig1 expression is indeed selectively down-regulated, though not completely abolished, in the cochlear column (arrow, Figure 5D; and data not shown). Interestingly, Rig1 expression is not affected in the anterior extramural migratory stream (Figure 5D), derived from the precerebellar Wnt1+lineage domain, where Hoxa2 regulates the expression of the slit receptor Robo2 [35]. Furthermore, Rig1 expression is not affected in Hoxb2DKOmutants (data not shown). Thus, our data show that Hoxa2 selectively regulates Rig1 expression during the guidance of contralateral AVCN projec-tions.
Abnormal specification and innervation of olivocochlear neurons in Hoxb1 and Hoxb2 mutant mice
The OC neurons become subdivided into medial and lateral components (MOC, LOC). The MOC neurons support OHC maturation at early postnatal stages and regulate the prestin-induced vibration of OHCs in the cochlea [12,21], and the LOC neurons, jointly with the MOC neurons, protect the cochlea from acoustic damage [22,31,32]. We next assessed whether the axonal behavior of efferent MOC neurons and synaptic MOC terminals on OHCs are affected in P8 and adult Hox mutant mice.
From E18.5 onwards, MOC motor axons have reached the contralateral cochlea. We thus injected DiI or dextran in Hoxb1null, Hoxb1lateCKO, Hoxb2DKO, or Hoxa2nullmutant cochleae, and in their respective controls, to retrogradely label the fluorescent bundle of MOC efferent fibers crossing the midline (Figure 6A). No crossing axons are identified in P8 Hoxb1null and Hoxb1lateCKO mutants (Figure 6B), in keeping with the lack of OC molecular markers observed at earlier stages (Figure S5). While the MOC bundle develops normally in E18.5 Hoxa2null mutant brains, no axons cross the midline in Hoxb2DKOmutants (Figure 6C), albeit a few presumptive OC Gata3+cells are detected at earlier stages (Figure S5C, S5F). This suggests that, either the few Hoxb2DKO mutant MOC neurons fail to target the cochlea, or that our axonal tracing
procedure is not sufficiently sensitive to label just a few crossing axons.
To further ascertain whether a few MOC axons may nonethe-less reach the cochlea and establish synaptic contact in Hoxb1 and Hoxb2 mutant animals, we used transmission electron microscopy and looked for MOC terminals contacting OHCs in the organ of Corti (Figure 6D). In contrast to WT animals, in which 1 to 2 MOC terminals are normally seen in synaptic contact with individual OHCs (n = 4; 54 MOC terminals on 32 OHCs; Figure 6E, 6F), almost no MOC terminals are found in adult
Hoxb1null cochleae (n = 4; 1 MOC terminal on 40 OHCs;
Figure 6E, 6F). Similarly, a few residual synaptic contacts are identified in adult Hoxb1lateCKO(n = 6; 12 MOC terminals on 64 OHCs; Figure 6E, 6F) and Hoxb2DKO mutant cochleae (data not shown), indicating that, although in highly reduced number, some MOC neurons are able to innervate OHCs in these mutant mice (Figure 6F).
Next, we investigated whether LOC neurons were properly specified in P8 Hoxb1 mutant mice. While ChAT and Tbx20 label the cholinergic population of LOC neurons, VGlut2 labels the glutamatergic population of the LSO, which is primarily derived from the Atoh1+lineage (Figure S7) [16]. No ChAT+or Tbx20+ cells are found in the LSO of Hoxb1nullbrains, whereas very few positive cells can be identified in Hoxb1lateCKO mutant brains (Figure S7B). On the contrary, vGlut2 expression is only slightly decreased, particularly in Hoxb1null mutants, possibly because the LSO is only partially derived from r4, as previously shown [3,16]. Thus, the populations primarily affected in Hoxb1 and Hoxb2 mutants are the cholinergic LOC neurons. Since the LSO largely forms within r5, this implies a previously unnoticed migration of some r4-derived cholinergic LOC cells into r5, possibly accom-panying in part the migration of FBM neurons.
In summary, our molecular and cellular data confirm the absence of MOC and LOC efferent neurons in Hoxb1nullmutants, but reveal the residual presence of a few MOC connections and some LOC neurons in Hoxb1lateCKOand Hoxb2DKOadult animals. This suggests that specification of OC neurons is primarily dependent on Hoxb1 expression in progenitor cells and on Hoxb2 expression in early post-mitotic neurons for their normal migratory and connectivity properties.
Abnormal morphology of cochlear hair cells in Hoxb1 and Hoxb2 mutant mice
The failure of MOC neurons to innervate OHCs and the absence of LOC neurons innervating IHCs might affect the correct development of cochlear hair cells and/or render them
more susceptible to degenerative acoustic trauma
[22,25,28,32,73]. To assess hair cell morphology in Hox mutant cochleae, we used scanning electron microscopy on the apical and basal turns of WT, Hoxb1null, Hoxb1lateCKOand Hoxb2DKOcochleae.
Since the medial efferent system is mature prior to the onset of hearing [21], we performed this analysis at P8, when the interactions of MOC fibers with OHCs become established, and in 3-month-old animals, when the centrifugal cochlear connections are fully functional (Figure 7 and Figure S8). In this way, we could discriminate a developmental intrinsic defect of OHCs from a defect due to the absence of MOC/OHCs interactions.
Hoxb2DKOversus Hoxb1null: p = 0.003). However, no statistically significant difference is found between Hoxb1lateCKOversus Hoxb2DKO(p = 0.39). (E)
Histogram showing the percentage of the VLL area size in WT and Hoxa2null. No statistically significant difference is found (WT versus Hoxa2null: p = 0.56). FBM, facial branchiomotor nucleus; VLL, nucleus of lateral lemniscus; IC, inferior colliculus; MG, medial geniculate nucleus. Scale bars, 200 mm. See also Figure S6.
Figure 4. The cochlear nuclear complex is differently affected inHoxb1,Hoxb2, andHoxa2mutants. (A) Ectopic YFP+r4-derived cells (arrows) are observed in the cochlear microneuronal shell (mnsh) (limited by solid line) of P8 Hoxb1nulland Hoxb1lateCKOmutant sagittal sections.
These cells now express Pax6 indicating that they are granule cells (white arrowheads). (B) Hoxb2 expression is decreased in the PVCN of Hoxb1nulland in Hoxb1lateCKOmutants (asterisks). On the contrary, Hoxa2 expression is increased and Atoh7, normally expressed at high levels only in the AVCN, is
dramatically up-regulated in the PVCN of P8 Hoxb1 (B) and Hoxb2DKOmutant pups (C) (arrows). Arrowheads in WT indicate Hoxa2 and Atoh7 low-Hox Genes in Central Auditory Circuits
Normally, three rows of OHCs and one row of IHCs are orderly arrayed along the entire organ of Corti at both ages. High magnification images of the hair bundles of individual WT OHCs illustrate the normal three rows of stereocilia of increasing height arranged in the characteristic V-shaped morphology; the latter appears slightly wider at the basal than apical turn (Figure 7 and Figure S8; n = 8). We found no obvious differences in the shape and organization of OHCs at the apical and basal cochlear turns in Hoxb1null(n = 6) and Hoxb1lateCKO(n = 4) pups at P8 (Figure 7A and Figure S8A), indicating a normal morphological development of the cochlea in young Hoxb1 mutant pups. However, when the architecture of the OHC area was assessed in 3-month-old animals, once the MOC/OHCs functional interactions are established and the cochlea has become fully responsive to sound, Hoxb1nulladult mice show severely disorganized OHC rows with occasional loss of hair cells in the apical turn (white arrowheads in Figure 7B) (Hoxb1null, n = 6: on average 7.2/100.8 OHCs are missing, versus WT, n = 8: 0/99 OHCs missing; Figure 7D). Furthermore, close observation of individual OHCs in Hoxb1null cochleae indicates that most stereocilia lose their typical V-shaped arrangement, as well as their organized structure and character-istic differences in ciliar length (arrows in Figure 7B). On the contrary, no IHCs are lost in these mutants, even if their cilia appear weakly disarranged (red arrowheads in Figure 7B). Only slight abnormalities in ciliar organization and orientation are observed in the basal turns of Hoxb1nullcochleae (arrows in Figure S8B; n = 6), indicating that the major defects occur predominantly at the cochlear apex. In contrast, in Hoxb1lateCKO and Hoxb2DKO cochleae, morphological defects of OHCs are less severe compared to those in Hoxb1nullmutants, although a loss of OHCs is still statistically significant, with occasionally missing cells (Hoxb1lateCKO, n = 6: average of 3.1/99.4 OHCs missing; Hoxb2DKO, n = 3: 1.3/100.1 OHCs missing; Figure 7D) and moderate OHC and IHC ciliar malformations (Figure 7B, 7C). In any case, the major abnormalities observed in Hoxb1 and Hoxb2 mutants are the severe morphological defects in ciliar shape and organization, rather than the OHC loss, which was negligible, even if statistically different between the genotypes. Finally, no abnormalities are observed in basal regions of Hoxb1lateCKOand Hoxb2DKOcochleae (Figure S8B; and data not shown).
Taken together, we found strong late postnatal morphological defects of outer hair cells in Hoxb1null mutants, particularly in the apical region of the cochleae where low-frequency sounds are normally perceived [11]. In addition, minor defects were detected in the ciliar shape and number of hair cells in Hoxb1lateCKOand Hoxb2DKOmutants, indicating that abnormal development of r4-derivatives in the auditory brainstem can affect, with different severities, the long-term survival and organization of cochlear hair cells.
Hearing loss in Hoxb1 and Hoxb2 mutant mice
The observed defects of the different components of the central auditory pathway leading to sound perception prompted us to test general auditory function in Hoxb1 and Hoxb2 mutant mice. We
measured the auditory brainstem response (ABR) in which a series of electrical potentials evoked by auditory stimuli ranging from 110 to 40 dB SPL (Sound Pressure Level) are analyzed, de-termining the lowest decibel level, or threshold, at which a response peak is reproducibly present [74]. The sequence of waves of the ABR reflects the synchronous short-latency synaptic activity of many neurons in successive nuclei along the central auditory pathway. We first analyzed the ABR response in control, Hoxb1null and Hoxb1lateCKOmice from 1 to 12 months of age (n = 307). While control mice have a normal 40 dB SPL threshold, the threshold is elevated to 90 dB SPL in Hoxb1nullmice representative ABRs of 3-month-old mice (Figure 8A). Hoxb1lateCKOmutant mice also show a pathologically elevated average threshold, although lower than that of Hoxb1nullmice (Figure 8A, 8C). In all ages examined, the threshold values of the responding Hoxb1 mutant mice are significantly higher than those of control mice (Figure 8C). Analyzing all data with regard to age, we observed a progressive increase of the hearing threshold for all three groups, although with different severities (doubled for Hoxb1null and 1.6 times for Hoxb1lateCKOwith respect to WT), most likely due to a secondary degeneration of cochlear hair cells and/or related afferent neural structures [75]. Interestingly, our data do not show any differences in the latencies of the evoked waves (Figure 8B), suggesting that the auditory stimuli can seemingly travel normally along the successive nuclei of the central auditory pathway, once the decibel levels surpass the elevated threshold. Furthermore, we analyzed a group of Hoxb2DKO3-month-old mutant mice (n = 5), together with their respective controls, and found an average threshold similar to that of Hoxb1lateCKOmice at the same age (Figure 8D).
Taken together, our data demonstrate that in the absence of Hoxb1 or Hoxb2 function, low-amplitude sounds are not perceived in young and adult mice.
Environmental auditory stimuli are not the primary cause of auditory impairment in Hoxb1nullmice
Previous studies have shown that the MOC and LOC neurons are required in protecting the cochlea against acoustic injury [22,28,31,32]. Thus, the lack of OC neurons observed in Hoxb1null mutant mice may render these mice particularly hypersensitive to environmental sounds and lead to severe OHC damage and hearing defects. To directly address this issue, a group of WT and Hoxb1null pups (n = 5) were placed in an acoustically isolated environment at birth, and their hearing capacity was tested after one month of age. We found that acoustically isolated Hoxb1null mice have the same threshold shift as non-acoustically isolated mice (Figure 8E). This suggests that the environment is not the primary cause of the auditory threshold defects observed in Hoxb1nullmice. However, the absence of protection from acoustic injury might determine secondarily the progressively more drastic increase of threshold observed with age in mutants compared to WT mice [76].
expressing regions. (D) Formation of the AVCN is strongly affected in E18.5 Hoxa2nullbrains, as seen by decreased expression of Atoh7 and Hoxb2
(arrows). (E) Summary schematic indicating that in the absence of Hoxb1 and Hoxb2, the PVCN (r4-derived in brown) has acquired AVCN-like features (r2/3-derived in yellow) and YFP+cell (brown) contribute to the shell. (F) The dorsal-most regions of WT and Hoxb1nullhindbrains at r3 and r4 levels on
adjacent coronal sections hybridized with Atoh1 and revealed by YFP epifluorescence (indicates r4 levels). Atoh1 is expressed in progenitors and differentiating cells migrating along the lateral ridge. The Atoh1-expressing domain is reduced in r4 compared to r3 in WT, whereas an enlarged Atoh1-expressing domain (arrowhead) is identified in r4 of Hoxb1nullembryos. (G) On adjacent coronal sections at E14.5, YFP+(r4-derived) cells located
more laterally, do not overlap with Atoh1+cells in the presumptive cochlear nucleus (CN), which originates from the r2–r5 auditory lip. In the absence of Hoxb1, YFP+cells invade the Atoh1+domain, thus acquiring the Atoh1 fate of adjacent rhombomeres. Cb, cerebellum; CES, caudal extramigratory stream. Scale bars, 200 mm (A up panels, B, C, D), 50 mm (A bottom panels), 100 mm (F, G). See also Figure S2.
Discussion
Rhombomere 4 contribution to the auditory system
Our present r4-restricted fate map has confirmed previous studies, but also highlighted specific aspects that were not recognized before [6,16]. In particular, we found that r4 contributes primarily to the generation and specification of auditory nuclei involved in sound transmission and amplifica-tion, as well as in the establishment of specific sensorimotor auditory circuitry during development (Figure 1M). Previous studies showed that ventral r4 is responsible for the specification of distinct subtypes of motor neurons, such as facial branchio-motor and inner ear efferent neurons [21,44,61,77]; here, we show that dorsal r4 contributes to alar-plate-derived sensory components, such as the VLL, PVCN and DCN. In particular, cells migrating rostrally from r4 along the lateral lemniscus tract form the VLL nucleus, whereas dorsal sensory cells remaining within r4 contribute massively to the CN complex (PVCN and DCN), as well as to the vestibular and trigeminal sensory columns (M.D., L.P., M.S., unpublished). Interestingly, we find that r4-derived cochlear sensory neurons form jointly with basal-plate-derived motor structures two distinct auditory sensorimotor feedback sub-circuits essential for proper hearing. Some PVCN neurons together with auditory efferent MOC neurons generated within r4 support the sound-evoked MOC reflex, which terminates directly on the OHCs of the cochlea and modulates the gain of the cochlear amplifier [23,24,25,26]. Interestingly, VCN neurons are also involved in the MEM reflex loop [29] through the action of the facial motor nucleus, originated in ventral r4 and strongly affected in Hoxb1 mutant mice [44,61] (data not shown). This implies that a single rhombomere, in this case r4, contributes to various derivatives of the auditory pathway; these are distributed across several rhombomeres via selective migrations and structurally linked into functional circuits essential for proper hearing.
We also found that the majority of r4-derivatives do not overlap with the r3/r5-derived Atoh1+ lineage, which contributes to the AVCN and to the nuclei of the superior olivary complex involved in the spatial localization of sounds [16,19]. Although further analyses are necessary to characterize the single populations derived from r4, our study suggests that r4 contributes more to inhibitory neurons (GABAergic and glycinergic) of the VLL and CN than to excitatory glutamatergic sub-populations. Firstly, we found that the VLL nucleus, which contains a majority of inhibitory Gad67+neurons, is mainly an r4-derivative, differently from the VGlut2+DLL, which is excitatory and originates primarily from the Atoh1+lineage [17,19,51]. Secondly, r4-derived rhombic lip cells do not contribute to the cochlear granule cell populations, nor to octopus and globular bushy cells, all of which are glutamatergic Atoh1-derivatives [15,53]. Thirdly, the change of r4 to r3 identity as a result of Hoxb1 inactivation in Hoxb1 and
Hoxb2 mutants, leads to an increase of the excitatory populations (such as the cochlear granule cell population) and decrease of the inhibitory cell types (such as the Gad67- and Gata3-expressing populations in the VLL). Fourthly, the Atoh7+ glutamatergic neurons, which derive from the Atoh1-expressing neuroepithelial regions, are massively increased in the PVCN of Hoxb1 mutant mice. Accordingly, mutant r4/YFP+neurons ectopically project to the MNTB nucleus, as normally done by AVCN cells originating from the Atoh1+ lineage in r3 [16,72]. Finally, we observed an increased dorsal r4 Atoh1+domain in Hoxb1nullmutants; this may be correlated to the ectopic production of glutamatergic granule cells and Atoh7+neurons in the mutant r4-derived CN.
We therefore propose that Hoxb1 is indirectly involved in regulating and/or modulating the ratio between inhibitory and excitatory neurons in the r4-derived auditory circuits. Since r4 is changed to a more rostral identity (r3) in absence of Hoxb1 function, the rhombomere-specific ratio between GABAergic/ glycinergic and glutamatergic neuronal fates might be conse-quently altered. Previous studies have shown that Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord and cerebellum [78,79,80], and is required for inhibitory GABAergic and glycinergic fate in the cochlear nucleus [15]; hence, it is plausible to speculate that Ptf1a expressed in the r4 ventricular zone may act downstream of, or together with Hoxb1 in the determination of r4-specific inhibitory features; altered Ptf1a expression may be responsible for the loss of GABAergic neurons in the VLL and/or for the ectopic expression of Atoh1, production of glutamatergic granule cells and Atoh7+neurons in the r4-derived CN.
However, some Atoh1+cells do derive normally from r4, even if the corresponding progenitor domain is reduced compared to that of other rhombomeres, and might thus also partially contribute to the glutamatergic lineage. Intersectional long-term fate mapping between r4 and neuronal subtype-specific mouse lines together with careful characterization of individual sub-populations will be required to fully elucidate this aspect.
Distinct regulation of Hoxb2 and Hoxa2 expression by Hoxb1 during patterning of sensory r4-derived neuronal structures
Previous studies reported that Hoxb1 together with Hoxb2 are crucial in impinging on an r4 identity during rhombomere patterning, and that ventral r4 is changed into a more anterior identity in the absence of Hoxb1, based on ectopic expression of
markers and abnormal behavior of FBM neurons
[34,37,38,44,61]. Here, we show that a similar regulation occurs also in dorsal r4 during the specification of auditory sensory derivatives. We found a re-patterning of r4 into r3 identity in Hoxb1 mutants, in which Hoxb1 is either constitutive (Hoxb1null) or conditionally (Hoxb1lateCKO) eliminated in r4, and in Hoxb2DKO
Figure 5. Abnormal cochlear connectivity inHoxb1,Hoxb2andHoxa2mutants. (A) Oblique P8 coronal sections show abnormal presence of YFP+fibers projecting to the medial nuclei of the trapezoid body (MNTB) in Hoxb1nulland Hoxb1lateCKOmutants. Right panels: enlarged views of the boxed areas to the left depicting abnormal YFP+terminals (arrowheads) of labeled crossed fibers (arrows) surrounding cells of the MNTB (limited by solid line). (B) Schematic representation showing the position of the injected dextran at the level of the PVCN (asterisk). Normally, PVCN interneurons innervate contralateral MOC neurons, as indicated in the control coronal section (arrow). In Hoxb2DKOmutants, projections originating from the PVCN target now AVCN-specific targets, such as the contralateral MNTB (cMNTB) (arrowhead) and the lateral superior olivary (LSO) nuclei. (C) Schematics summarizing the normal connectivity pattern of cochlear AVCN neurons towards the nuclei of the superior olivary complex (SOC) complex, and the abnormal presence of YFP+neurons behaving like AVCN neurons when Hoxb1 or Hoxb2 are inactivated. (D) A subpopulation of AVCN-labeled axons project abnormally to the ipsilateral MNTB (see arrowhead) when Hoxa2 is conditionally inactivated in the Wnt1+(rhombic lip) domain. Expression of the Slit receptor Rig1 (or Robo3) is decreased in the VCN, indicating that absence of Rig1 affects midline crossing of AVCN projections. (E) Schematics summarizing the abnormal axonal projections of AVCN neurons in Wnt::Cre;Hoxa2lox/lox mice after dextran injection in the AVCN. AVCN, anterior ventral cochlear nucleus; PVCN, posterior ventral cochlear nucleus; PVCN IN, PVCN interneurons; MOC, medial olivocochlear neurons; aes, anterior extramural stream. Scale bars: 400 mm (A left panels, B), 50 mm (A right panels).
mice, in which Hoxb1 expression fails to be maintained in r4. In the absence of Hoxb1, Hoxb2 expression is reduced and Hoxa2 expression is increased in r4 at levels similar to those in r3, leading ultimately to the loss of specific r4-derived auditory nuclei (VLL and PVCN) and the ectopic formation of r3-like derived structures (AVCN and cochlear granule cells).
We thus conclude that within this sensory system Hoxb1 and Hoxb2 are critically required during the specification of r4-derived structures, and that in their absence, r4 alar and rhombic lip derivatives largely acquire r3-like features. This is based on our data and is consistent with previous intersectional fate mapping
Figure 6. Affected connectivity of medial olivocochlear (MOC) neurons inHoxb1 and Hoxb2mutant mice. (A) Schematic view of a brain coronal section indicates the insertion of a DiI or dextran crystal into the cochlea to label controlateral MOC neurons. (B) Retrogradely-labeled MOC axons normally project across the midline as a compact bundle (arrow). No MOC axons crossing the midline are retrogradely labeled in Hoxb1nulland Hoxb1lateCKObrains (asterisks). (C) Similarly, MOC fibers fail to cross the midline in Hoxb2DKOmutant brains (asterisk), whereas no obvious defect is observed in Hoxa2nullmutants (arrow). (D) Schematic view of the organ of Corti, showing terminal innervation of the OHCs by the MOC efferent
neurons (arrows). (E) Transmission electron microscopy of OHCs in adults WT, Hoxb1nulland Hoxb1lateCKOcochleae. In high magnification views, MOC terminals synapse on OHCs in WT and Hoxb1lateCKOcochleae, even if at much reduced ratio (F); a sub-synaptic cisterna is visualized inside the OHC
(arrowheads). No MOC terminals are detected in Hoxb1nullcochleae. (F) Histogram showing the ratio of the number of MOC synaptic contacts on OHC visualized in TEM experiments in controls, Hoxb1nulland Hoxb1lateCKOmutants. MOC/OHCs ratio: WT (n = 3; 32 OHCs): 1.960.2; Hoxb1lateCKO(n = 6; 64
OHCs): 0.260.2; Hoxb1null(n = 4; 40 OHCs): 0.160.1. Inter-genotype ANOVA p,0.001; Hoxb1lateCKOversus WT: p,0.001; Hoxb1nullversus WT: p,0.001. Scale bars, 400 mm (B, C), 10 mm (E, left panel of WT), 50 mm (E, left panels of Hoxb1nulland Hoxb1lateCKO), 1 mm (E, right panels). See also Figures S5
and S7.
doi:10.1371/journal.pgen.1003249.g006
Figure 7. Late degeneration of OHCs in the apical turn ofHoxb1andHoxb2mutant cochleae. (A) Scanning electron microscopy (SEM) views of the cochlea at P8: an overview of the apical turns of WT, Hoxb1nulland Hoxb1lateCKOcochleae showing three orderly arrayed rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs). Representative high magnification images illustrate stereocilia of hair bundles of single OHCs arranged according to their different lengths. Shape and organization of OHCs in apical regions are normal at this stage in both mutants. (B) SEM views of 3-month-old WT, Hoxb1nulland Hoxb1lateCKOcochleae and representative higher magnification images of OHCs. In Hoxb1nulland Hoxb1lateCKO
cochleae, OHCs have lost their regular organization and fail to develop in some areas (white arrowheads). Moreover, in Hoxb1nullcochleae most stereocilia have completely lost their typical V-shaped morphology and their characteristic differences in lengths (arrows). OHCs are less severely affected in Hoxb1lateCKOcochleae. IHC cilia appeared weakly disarranged (red arrowheads). (C) SEM views of 3-months-old WT and Hoxb2DKOmutant cochleae and higher magnifications of representative OHCs. Note that, similarly to Hoxb1lateCKOcochleae, Hoxb2 mutants have occasional missing
OHCs (white arrowheads), disarranged IHC cilia (red arrowheads) and disorganized OHC stereocilia (arrows). (D) Histogram quantifying the percentage of OHC loss in controls, Hoxb1null, Hoxb1lateCKOand Hoxb2DKOcochleae. While controls (n = 8) showed no OHC loss, in Hoxb1nullmutants (n = 6) 7.260.8% of OHCs were absent, whereas 3.160.9% and 1.360.5% were lost in Hoxb1lateCKO(n = 6) and Hoxb2DKO(n = 3) cochleae, respectively.
Inter-genotype ANOVA p,0.001; Hoxb1nullversus WT: p,0.001; Hoxb1lateCKOversus WT: p,0.001; Hoxb2DKOversus WT: p = 0.02. Scale bars, 10 mm (A, B, C, left panels), 1 mm (A, B, C, right panels). See also Figure S8.
with r2 and r3/r5-specific enhancers together with the selective inactivation of Atoh1 in r3 and r5 [6,16].
In addition, we observed that Hoxb2DKO and Hoxb1lateCKO reproduce a similar phenotype. Indeed, although early Hoxb1 expression is able to partially specify r4 identity in Hoxb1lateCKOand Hoxb2DKO mutants, failure to maintain Hoxb1 at later stages inhibits further development of r4-derived structures, leading ultimately to a milder phenotype when compared to constitutive Hoxb1 null mutants. We observed slight differences in the phenotypic severity of these two mutant lines, which might essentially be due to the differences in timing of Hoxb1 inactivation in r4.
Importantly, our data show that in r4 Hoxb2, besides being involved in maintaining high levels of Hoxb1 in progenitor cells (our study and [37,58]), also plays a key role in relaying
r4-dependent regional fate to post-mitotic cells. In support of this, we show that Hoxb2 is expressed in both r4-derived progenitors and post-mitotic neurons, and that its expression is maintained in r4-derived structures, such as the VLL, VCN and OC.
A different situation applies for Hoxa2, which plays crucial roles in cell migration and axonal connectivity during hindbrain development, and whose expression is also maintained in the auditory sensory nuclei [36,66]. Hoxb1 expression is not affected in Hoxa2nullmutants, as previously reported [66] and, accordingly, no defects were observed in VLL and MOC development, two r4-derived structures. However, we found that Hoxa2 expression in r4 is increased in Hoxb1 and Hoxb2DKOembryos, both in progenitors and, importantly, in post-mitotic cells. This ectopic expression is maintained in the postnatal PVCN of these mutants together with
Figure 8. Elevated thresholds of auditory brainstem responses (ABR) in Hoxb1 andHoxb2 mutant mice. (A) Representative ABR measurements of 3 month-old WT, Hoxb1nulland Hoxb1lateCKOmice. Five ABR peaks are the normal sequential responses evoked by an auditory
stimulus at the level of the auditory nerve and along the series of auditory nuclei of the brainstem. The threshold, the lowest intensity of sound at which the response is present, is higher in Hoxb1 mutants than WT mice. (B) Latencies, the time intervals between the stimulus and the diverse response peaks, are normal at different intensities of sound. (C) Mean (6 SE) thresholds measured in dB SPL (Sound Pressure Level) for WT, Hoxb1lateCKOand Hoxb1nullmice at 1, 3, 6, 9 and 12 months of age. The differences between WT, Hoxb1lateCKOand Hoxb1nullgroups are statistically
significant at all ages (1 month: WT, n = 24, 41.260.5; Hoxb1null, n = 25, 79.062.1; Hoxb1lateCKO, n = 23, 58.362.8; 3 months: WT, n = 28, 41.260.4; Hoxb1null, n = 23, 84.361.7; Hoxb1lateCKO, n = 33, 63.062.4; 6 months: WT, n = 22, 45.262.5; Hoxb1null, n = 17, 96.562.6; Hoxb1lateCKO, n = 25, 72.862.7; 9
months: WT, n = 20, 51.262.5, Hoxb1null, n = 14, 102.163.1, Hoxb1lateCKO, n = 17, 77.963.1; 12 months: WT, n = 10, 5664, Hoxb1null, n = 12, 108.963.0,
Hoxb1lateCKO, n = 14, 82.164.6; ANOVA p,0.001; post hoc t-test p,0.001; WT versus Hoxb1null; WT versus Hoxb1lateCKO: p,0.001 for all stages). A progressive increase of hearing thresholds in all groups with age is more prominent in Hoxb1nulland Hoxb1lateCKOmutants than in the control mice.
(D) Mean (6 SE) thresholds of ABR for 3-month-old control and Hoxb2DKOmice. The differences between the two groups are statistically significant (WT, n = 5, 5261.3; Hoxb2DKO, n = 5, 6965.7; t-test p = 0.013); the threshold increase is similar to that of Hoxb1lateCKOmice at the same age. (E) ABR of 1
month-old WT and Hoxb1nullmice put in an acoustically isolated environment from birth. Auditory thresholds are increased (WT: 48.960.2; Hoxb1null: 84.260.4; p,0,01) similarly to those of non-acoustically isolated mutants (C). dB, decibel.
doi:10.1371/journal.pgen.1003249.g008
